The following abbreviations are used throughout this application:
A. cal.--Autographa californica PA0 AcMNPV--Autographa californica nuclear polyhedrosis virus PA0 AaIT--Androctonus australis insect toxin PA0 bp--base pairs PA0 CPU--contractile paralysis unit PA0 ECV--extracellular virus PA0 GV--granulosis virus PA0 kD--kilodaltons PA0 LT--lethality time PA0 MOI--multiplicity of infection PA0 NPV--nuclear polyhedrosis virus PA0 OB--occlusion body PA0 Occ---occlusion negative virus(es) PA0 Occ+--occlusion positive virus(es) PA0 PCR--polymerase chain reaction PA0 PDV--polyhedron derived virus PA0 PFU--plaque forming unit PA0 p.i.--post-infection PA0 PIB--polyhedron inclusion body (also known as OB) PA0 ST--survival time
The family of DNA insect viruses known as Baculoviridae includes nuclear polyhedrosis viruses (NPV) and granulosis viruses (GV). These viruses produce occlusion bodies (OBs) in their life cycle. Also included are the non-occluded viruses (NOV), which do not produce OBs in their life cycle. Another family of DNA insect viruses are the entomopox viruses.
Over 400 baculoviruses have been identified as present in invertebrates. Examples of NPVs include Lymantria dispar NPV (gypsy moth NPV), Autographa californica MNPV, Syngrapha falcifera NPV (celery looper NPV), Spodoptera litturalis NPV, Spodoptera frugiperda NPV, Heliothis armigera NPV, Mamestra brassicae NPV, Choristoneura fumiferana NPV, Trichoplusia ni NPV, Helicoverpa zea NPV, etc. Examples of GVs include Cydia pomonella GV (codling moth GV), Pieris brassicae GV, Trichoplusia ni GV, etc. Examples of NOVs are Orcytes rhinoceros NOV and Hleiothis zea NOV. Examples of entomopox viruses include Melolontha melonotha EPV, Amsacta moorei EPV, Locusta migratoria EPV, Melanoplus sanguinipes EPV, Schistocerca gregaria EPV, Aedes aegypti EPV, Chironomus luridus EPV, etc.
The use of baculoviruses and entomopox viruses as bioinsecticides holds great promise. One of the major impediments to their widespread use in agriculture is the time lag between initial infection of the insect and its death. This lag can range from a few days to several weeks. During this lag, the insect continues to feed, causing further damage to the plant. A number of researchers have attempted to overcome this drawback by inserting a heterologous gene into the viral genome, so as to express an insect controlling or modifying substance, such as a toxin, neuropeptide or an enzyme (Bibliography entries 1-5).
The life cycle of baculoviruses, as exemplified by AcMNPV, includes two stages. Each stage of the life cycle is represented by a specific form of the virus: Extracellular viral particles (ECV) which are nonoccluded, and occluded virus particles (OB) (6,7). The extracellular and occluded virus forms have the same genome, but exhibit different biological properties. The maturation of each of the two forms of the virus is directed by overlapping sets of vital genes, some of which are unique to each form.
In its naturally occurring insect infectious form, multiple virions are found embedded in a paracrystalline protein matrix known as an occlusion body (OB), which is also referred to as a polyhedron inclusion body (PIB). The proteinaceous vital occlusions are referred to as polyhedra (polyhedron is the singular term). A polyhedrin protein, which has a molecular weight of 29 kD, is the major viral-encoded structural protein of the vital occlusions (6,8). (Similarly, GVs produce OBs which are composed primarily of granulin, rather than polyhedrin).
The viral occlusions are an important part of the natural baculovirus life cycle, providing the means for horizontal (insect to insect) transmission among susceptible insect species. In the environment, a susceptible insect (usually in the larval stage) ingests the viral occlusions from a contaminated food source, such as a plant. The crystalline occlusions dissociate in the gut of the susceptible insects to release the infectious vital particles. These polyhedron derived viruses (PDV) invade and replicate in the cells of the midgut tissue (6).
It is believed that virus particles enter the cell by endocytosis or fusion, and the viral DNA is uncoated at the nuclear pore or in the nucleus. Viral DNA replication is detected within six hours. By 10-12 hours post-infection (p.i.), secondary infection spreads to other insect tissues by the budding of the extracellular virus (ECV) from the surface of the cell. The ECV form of the virus is responsible for cell to cell spread of the virus within an individual infected insect, as well as transmitting infection in cell culture.
Late in the infection cycle (12 hours p.i.), polyhedrin protein can be detected in infected cells. It is not until 18-24 hours p.i. that the polyhedrin protein assembles in the nucleus of the infected cell and virus particles become embedded in the proteinaceous occlusions. Viral occlusions accumulate to large numbers over 4-5 days as cells lyse. These polyhedra have no active role in the spread of infection in the larva. ECVs in the haemolymph multiply and spread, leading to the death of the larva (6-8).
When infected larvae die, millions of polyhedra remain in the decomposing tissue, while the ECVs are degraded. When other larvae are exposed to the polyhedra, for example, by ingestion of contaminated plants or other food material, the cycle is repeated (6).
In summary, the occluded form of the virus is responsible for the initial infection of the insect through the gut, as well as the environmental stability of the virus. PDVs are essentially not infectious when administered by injection, but are highly infectious orally. The non-occluded form of the virus (i.e., ECV) is responsible for secondary and cell to cell infection. ECVs are highly infectious for cells in culture or internal insect tissues by injection, but essentially not infectious by oral administration.
The use of recombinant baculoviruses expressing foreign proteins which are toxic to insects is facilitated by the fact that these viruses are not pathogenic to vertebrates or plants. In addition, the baculoviruses generally have a narrow host range. Many strains are limited to one or a few insect species.
The Autographa californica nuclear polyhedrosis virus (AcMNPV) is the prototype virus of the family Baculoviridae. The AcMNPV virus was originally isolated from Autographa californica (A. cal.), a lepidopteran noctuid (which in its adult stage is a nocturnal moth), commonly known as the alfalfa looper. This virus infects 12 families and more than 30 species within the order of Lepidoptera insects (9). It is not known to infect productively any species outside this order. The most widely studied baculovirus is AcMNPV. This virus utilizes many of the protein maturation and transport systems that occur in higher eukaryotic cells.
In this invention, a gene coding for an insect controlling protein is inserted into a suitable location in the viral genome. Heterologous genes inserted into AcMNPV produce proteins which are biologically active in the infected insect cells. These proteins, for the most part, undergo post-translational processing to produce and secrete recombinant products very similar, if not identical, to those of authentic proteins. A protein thus expressed enhances the bioinsecticidal effect of the virus.
One such protein is the toxin designated AaIT, which is produced by the venom of the North African scorpion Androctonus australis Hector. The toxin is 70 amino acids in length and binds to sodium channels in insects and causes contractile paralysis at the nanogram to picogram range in insect larvae. Because AaIT does not bind to mammalian sodium channels, AaIT is a candidate for use as a bioinsecticide to protect crops ingested by humans.
The region upstream of the coding region of the AaIT gene includes a signal sequence which directs the secretion of AaIT from the cell. Specifically, the signal sequence directs the toxin through the secretory pathway to the cell surface where it is secreted from the cell. During transport, enzymes cleave the signal sequence, leaving the mature AaIT.
There is a continuing need for genetically engineered recombinant insect viruses which express heterologous toxins in infected hosts. Infection by these recombinant viruses increases the speed of kill when compared with the wild-type virus.